Project Summary Extracellular vesicles (EVs) are cell-derived membranous structures carrying transmembrane proteins and luminal cargo including soluble cytoplasmic proteins and nucleic acids. They are a heterogeneous group of particles generally classified according to subcellular origin, dimension, and tetraspanin surface expression. Circulating EVs may act as nanocontainers capable of recognizing target cells through membrane receptors and communicating cargo through membrane fusion, a notoriously low probability process. Interaction with recipient cells can lead to their acquisition of EV surface proteins and luminal contents although the mechanisms controlling this exchange remain unclear. Their complex cargo and well as surface markers are highly variable with respect to functional impact and dependent upon the tissue context from which they are released. A central focus of the proposed work is the isolation and identification of functionally competent small EVs (30-150 nm) carrying an ion channel of interest which can then be transferred to a nave, non-expressing cell. Functionally significant EV-mediated channel translocation from one cell to another would require a high density of channel expression per vesicle during EV biosynthesis in the donor cell and reliable fusion with the plasma membrane and endocytic/phagocytic compartments of recipient cells. In order to facilitate EV membrane fusion we will co-express the viral protein fusogen hemagglutinin subtype 7 (HA). Expression of a genetically engineered blue light-activated Ca2+ channel switch (BACCS) which opens the Ca2+ selective ORAI ion channel in response to light will be used in the design of screening strategies exploring vesicle fusion and channel transfer. The light- sensitive probe will be used as screening tool for functional Ca2+ channel transfer in non-responsive recipient cells via isolated EVs derived from BACCS-ORAI expressing cells. Once we have established optimal conditions for EV-mediated channel transfer, we will leverage our experience in the study of the anion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR) in murine alveolar macrophages (AMs) and epithelial cells and study the EV-mediated transfer of CFTR to cftr-/- cells in the context of the disease of cystic fibrosis (CF) using adoptive transfer techniques.